The invention relates to phosphatases and more in specific to (genetically) modified phosphatases, pharmaceutical compositions comprising (genetically) modified phosphatases and the use of (genetically) modified phosphatases for treating or curing for example sepsis, inflammatory bowel disease or other inflammatory disease, or renal failure. The invention further relates to a method for producing phosphatases.
A phosphatase is an enzyme that dephosphorylates its substrate; i.e. it hydrolyses phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group. This action is directly opposite to that of phosphorylases and kinases, which attach phosphate groups to their substrates by using energetic molecules like ATP. Phosphatases can be categorised into two main categories: Cysteine-dependent Phosphatases (CDPs) and metallo-phosphatases. The latter ones are dependent on the presence of one or more metal ions in their active site(s) for activity.
CDPs catalyse the hydrolysis of a phosphoester bond via a phospho-cysteine intermediate. The free cysteine nucleophile forms a bond with the phosphorus atom of the phosphate moiety, and the P—O bond linking the phosphate group to the tyrosine is protonated, either by a suitably positioned acidic amino acid residue or a water molecule. The phospho-cysteine intermediate is then hydrolysed by another water molecule, thus regenerating the active site for another dephosphorylation reaction.
Metallo-phosphatases co-ordinate 1 or more catalytically essential metal ion(s) within their active site. There is currently some confusion of the identity of these metal ions, as successive attempts to identify them yield different answers. There is currently evidence that these metals could be Magnesium, Manganese, Iron, Zinc, or any combination thereof. It is thought that a hydroxyl ion bridging the two metal ions takes part in nucleophilic attack on the phosphate group
Phosphatases act in opposition to kinases/phosphorylases, which add phosphate groups to proteins. The addition of a phosphate group may activate or de-activate an enzyme (e.g., Kinase signalling pathways) or enable a protein-protein interaction to occur (e.g., SH3 domains); therefore phosphatases are integral to many signal transduction pathways. It should be noted that phosphate addition and removal do not necessarily correspond to enzyme activation or inhibition, and that several enzymes have separate phosphorylation sites for activating or inhibiting functional regulation. CDK, for example, can be either activated or deactivated depending on the specific amino acid residue being phosphorylated. Phosphates are important in signal transduction because they regulate the proteins to which they are attached. To reverse the regulatory effect, the phosphate is removed. This occurs on its own by hydrolysis, or is mediated by protein phosphatases.
Without limiting the present invention, alkaline phosphatases are discussed in more detail as an example of the herein described and claimed phosphatases. Alkaline phosphatase (ALP) (EC 3.1.3.1) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment.
Alkaline phosphatase has become a useful tool in molecular biology laboratories, since DNA normally possesses phosphate groups on the 5′ end. Removing these phosphates prevents the DNA from ligation (the 5′ end attaching to the 3′ end of the same or another molecule); also, removal of the phosphate groups allows radiolabeling (replacement by radioactive phosphate groups) in order to measure the presence of the labeled DNA through further steps in the process or experiment. For these purposes, the alkaline phosphatase from shrimp is the most useful, as it is the easiest to inactivate once it has done its job.
Another important use of alkaline phosphatase is as a label for enzyme immunoassays.
Moreover, alkaline phosphatases are used in the treatment of for example sepsis, inflammatory bowel disease, or renal failure.
Although the presently available (alkaline) phosphatases are useful in both diagnostics and disease treatment there is a need for alternative phosphatases with for example an altered (for example improved) specific activity, stability (for example in vivo T1/2, or stability in respect of storage (shelf-life)) or substrate specificity. Moreover, there is also a need for phosphatases with a different pH or temperature or salt (in)dependency profile.